Described herein are methods for identifying and preparing high affinity nucleic acid ligands that bind to a VEGF receptor. The method utilized herein for identifying such nucleic acid ligands is called SELEX, an acronym for Systematic Evolution of Ligands by EXponential Enrichment. This invention includes high affinity nucleic acids to a VEGF receptor. Further disclosed are RNA ligands to a VEGF receptor. Also included are oligonucleotides containing nucleotide derivatives modified at the 2xe2x80x2 position of the pyrimidines. Additionally disclosed are ligands to a VEGF receptor containing 2xe2x80x2-F modifications of the pyrimidines. This invention also includes high affinity nucleic acid inhibitors of VEGF signaling. The oligonucleotide ligands of the present invention are useful in any process in which binding of VEGF to a VEGF receptor is required. This includes, but is not limited to, their use as pharmaceuticals, diagnostics, imaging agents, and immunohistochemical reagents.
Angiogenesis in Disease
The growth of new blood vessels from existing endothelium (angiogenesis) is tightly controlled in healthy adults by opposing effects of positive and negative regulators. Under certain pathological conditions, including proliferative retinopathies, rheumatoid arthritis, psoriasis and cancer, positive regulators prevail and angiogenesis contributes to disease progression (reviewed in Folkman (1995) Nature Med. 1:27-31). In cancer, the notion that angiogenesis represents the rate limiting step of tumor growth and metastasis (Folkman (1971) New Engl. J. Med. 285:1182-1186) is now supported by considerable experimental evidence (reviewed in Aznavoorian et al. (1993) Cancer 71:1368-1383; Fidler and Ellis (1994) Cell 79:185-188; Folkman (1990) J. Natl. Cancer Inst. 82:4-6). The quantity of blood vessels in tumor tissue is a strong negative prognostic indicator in breast cancer (Weidner et al. (1992) J. Natl. Cancer Inst. 84:1875-1887), prostate cancer (Weidner et al. (1993) Am. J. Pathol. 143:401-409), brain tumors (Li et al.(1994) Lancet 344:82-86), and melanoma (Foss et al.(1996) Cancer Res. 56:2900-2903).
VEGF Signaling in Angiogenesis
A number of angiogenic growth factors have been described to date among which vascular endothelial growth factor (VEGF) appears to play a key role as a positive regulator of physiological and pathological angiogenesis (reviewed in Brown et al. (1997) in Control of Angiogenesis (Goldberg and Rosen, eds.), Birkhauser, Basel: 233-269; Thomas (1996) J. Biol. Chem. 271:603-606; Neufeld et al. (1999) FASEB J. 13: 9-22). VEGF is a secreted disulfide-linked homodimer that selectively stimulates endothelial cells to proliferate, migrate, and produce matrix-degrading enzymes (Conn et al. (1990) Proc. Natl. Acad. Sci.
U.S.A. 87: 1323-1327; Ferrara and Henzel (1989) Biochem. Biophys. Res. Commun. 161: 851-858; Gospodarowicz et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 7311-7315; Pepper et al. (1991) Biochem. Biophys. Res. Commun. 181: 902-906; Unemori et al. (1992) J. Cell. Physiol. 153: 557-562), all of which are processes required for the formation of new vessels. In addition to being the only known endothelial cell specific mitogen, VEGF is unique among angiogenic growth factors in its ability to induce a transient increase in blood vessel permeability to macromolecules (hence its original and alternative name, vascular permeability factor) (Dvorak et al.(1979) J. Immunol. 122:166-174; Senger et al.(1983) Science 219:983-985; Senger et al.(1986) Cancer Res. 46:5629-5632). Increased vascular permeability and the resulting deposition of plasma proteins in the extravascular space assists the new vessel formation by providing a provisional matrix for the migration of endothelial cells (Dvorak et al.(1995) Am. J. Pathol. 146:1029-1039). Hyperpermeability is indeed a characteristic feature of new vessels, including those associated with tumors (Dvorak et al.(1995) Am. J. Pathol. 146:1029-1039). Furthermore, compensatory angiogenesis induced by tissue hypoxia is now known to be mediated by VEGF (Levy et al.(1996) J. Biol. Chem. 271:2746-2753); Shweiki et al. (1992) Nature 359:843-845).
VEGF is produced and secreted in varying amounts by virtually all tumor cells (Brown et al. (1997) in Control of Angiogenesis, ibid). Direct evidence that VEGF and its receptors contribute to tumor growth was recently obtained by a demonstration that the growth of human tumor xenografts in nude mice could be inhibited by neutralizing antibodies to VEGF (Kim et al. (1993) Nature 362:841-844), by the expression of dominant-negative VEGFR2 (Millauer et al. (1996) Cancer Res. 56:1615-1620; Millauer et al. (1994) Nature 367:576-579), by low molecular weight inhibitors of VEGF receptor inhibitors (Strawn et al. (1966) Cancer Res. 56:3540-3545), or by the expression of antisense sequence to VEGF mRNA (Saleh et al. (1996) Cancer Res. 56:393401). Importantly, the incidence of tumor metastases was also found to be dramatically reduced by VEGF antagonists (Asano et al. (1995) Cancer Res. 55, 5296-5301; Warren et al. (1995) J. Clin. Invest. 95: 1789-1797; Claffey et al. (1996) Cancer Res. 56:172-181; Melnyk et al. (1996) Cancer Res. 56, 921-924). Inhibitors of VEGF signaling may thus have broad clinical utility as anticancer agents. In addition to cancer, as noted above, other proliferative diseases characterized by excessive neovascularization such as psoriasis, age-related macular degeneration, diabetic retinopathy and rheumatoid arthritis could be treated with antagonists of VEGF signaling.
VEGF occurs in several forms (VEGF-121, VEGF-145, VEGF-165, VEGF-189, VEGF-206) as a result of alternative splicing of the VEGF gene that consists of eight exons (Houck et al. (1991) Mol. Endocrin. 5:1806-1814; Tischer et al. (1991) J. Biol. Chem. 266:11947-11954; Poltorak et al. (1997) J. Biol. Chem. 272: 7151-7158). The three smaller forms are diffusable while the larger two forms remain predominantly localized to the cell membrane as a consequence of their high affinity for heparin. VEGF-165 and VEGF-145 also bind to heparin (as a consequence of containing basic exon 7- and exon 6-encoded domains, respectively), albeit with somewhat lower affinity compared with VEGF-189 (that contains both exons 6 and 7). VEGF-165 appears to be the most abundant form in most tissues (Houck et al. (1991) Mol. Endocrinol., ibid; Carmeliet et al. (1999) Nature Med. 5: 495-502). VEGF-121, the only alternatively spliced form that does not bind to heparin, appears to have a somewhat lower affinity for the receptors (Gitay-Goren et al. (1996) J. Biol. Chem. 271:5519-5523) as well as lower mitogenic potency (Keyt et al. (1996) J. Biol. Chem. 271:7788-7795).
VEGF Receptors
Biological effects of VEGF are mediated by two homologous tyrosine kinase receptors, Flt-1 (VEGFR1) and Flk-1/KDR (VEGFR2) whose expression is highly restricted to cells of endothelial origin (de Vries et al. (1992) Science 255: 989-991; Millauer et al. (1993) Cell 72: 835-846; Terman et al. (1991) Oncogene 6: 519-524). Both receptors have an extracellular domain consisting of seven IgG-like domains, a transmembrane domain and an intracellular tyrosine kinase domain. The affinity of VEGFR1 for VEGF (Kd=1-20 pM) is higher compared to that of VEGFR2 (Kd=50-770 pM) (Brown et al. (1997) in Regulation of Angiogenesis, ibid; de Vries et al. (1992) Science 255, 989-991; Terman et al. (1992) Biochem. Biophys. Res. Commun. 187, 1579-1586). In human umbilical cord endothelial cells (HUVECs) in 2-dimensional culture, VEGFR2 is by far the more abundant receptor (Brown et al. (1997) in Regulation of Angiogenesis, ibid). In vivo, however, in quiescent endothelial cells, both receptors are expressed at low levels (Kremer et al. (1997) Cancer Res. 57, 3852-3859; Barleon et al. (1997) Cancer Res. 57: 5421-5425).
Both receptors are substantially upregulated when endothelial cells are activated by a variety of stimuli. Hypoxia, for example, induces an increase in expression of both VEGFR1 and VEGFR2 in endothelial cells (Tuder et al. (1995) J. Clin. Invest. 95: 1798-1807; Gerber et al. (1997) J. Biol. Chem. 272: 23659-23667; Brogi et al. (1996) J. Clin. Invest. 97: 469-476; Kremer et al. (1997) Cancer Res., ibid). For VEGFR 1, hypoxia leads to both direct activation via the flt-1 promoter that contains the hypoxia-inducible-factor-1 (HIF-1) consensus binding site (Gerber et al. (1997) J. Biol. Chem., ibid) and indirect activation via hypoxia-induced VEGF (Barleon et al. (1997) Cancer Res., ibid). VEGF-induced upregulation of VEGFR1 is mediated by both VEGFR1 and VEGFR2 (Barleon et al. (1997) Cancer Res., ibid). VEGFR2 is upregulated by VEGF (through VEGFR2 but not VEGFR1 ) (Kremer et al. (1997) Cancer Res., ibid; Wilting et al. (1996) Dev. Biol. 176:76-85) and possibly by a yet unidentified factor in hypoxia-conditioned media from myoblasts (Brogi et al. (1996) J. Clin. Invest., ibid). The expression of VEGFR2 in endothelial cells is also upregulated by bFGF and this accounts in part for the synergistic activation of endothelial cells by VEGF and bFGF (Pepper et al. (1998) Exp. Cell Res. 241, 414-425). In addition, since both kdr and flt-1 promoters contain a cis-acting fluid shear-stress-responsive element, VEGFR1 and VEGFR2 expression may be sensitive to variations in blood flow (Tuder et al. (1995) J. Clin. Invest., ibid).
Experiments using porcine aortic endothelial (PAE) cells transfected with the flt-1 or kdr receptor genes have suggested that VEGFR2 is the primary transducer in endothelial cells of VEGF-mediated signals related to changes in cell morphology and mitogenicity (Waltenberger et al. (1994) J. Biol. Chem. 269, 26988-26995). In the same study, stimulation of flt-1-transfected PAE cells with VEGF did not appear to produce detectable changes. More recently, however, it was demonstrated that VEGF signaling through VEGFR1 induces migration of monocytes and upregulation of tissue factor expression in both endothelial cells and monocytes (Clauss et al. (1996) J. Biol. Chem. 271, 17629-17634; Barleon et al. (1996) Blood 87, 3336-3343). Based on the observation that the extracellular domain of VEGFR2 is retained on a cation exchange resin only in the presence of VEGFR1 and that the VEGFR2 retention is enhanced when both VEGFR1 and VEGF were present, Kendall et al. have concluded that the two receptors have some affinity for one another and that this interaction is stabilized by VEGF (Kendall et al. (1996) Biochem Biophys. Res. Commun. 226, 324-328). When both receptors are expressed on cell surface, it appears likely that the VEGFR1/R2 heterodimer constitutes at least a fraction of the binding-competent VEGF receptor.
Gene Deletion Studies of VEGF and VEGF Receptors
The functions of VEGFR1 and VEGFR2 have further been elucidated by targeted gene deletion studies. While deletion of either VEGFR1 or VEGFR2 results in embryonic lethality as a result of vascular abnormalities, there are important differences in the two phenotypes.
In mice deficient in VEGFR1, endothelial cells are formed but organize into distended and dilated vessels (Fong et al. (1995) Nature 376: 66-70). Interestingly, mice that only lack the tyrosine kinase domain of VEGFR1 (and thus display the receptor on cell surfaces that is incapable of signaling) are viable, with the only detectable abnormality being the strongly suppressed macrophage migration in response to VEGF (Hiratsuka et al. (1998) Proc. Natl. Acad. Sci. 95: 9349-9354). Since vascular abnormalities of VEGFR1 knockout mice are similar to those observed in transgenic mice that overexpress VEGF during development, it has been suggested that VEGFR1 is primarily a negative regulator of VEGF signaling, and that partial inhibition of VEGF signaling is essential for proper vessel development (Hiratsuka et al. (1998) Proc. Natl. Acad. Sci., ibid). It is relevant to note in this context that VEGFR1 also exists as an alternatively spliced secreted extracellular domain that acts as a potent inhibitor of VEGF (Kendall et al. (1993) Proc. Natl. Acad. Sci., U.S.A. 90: 10705-10709). The importance of tightly controlled VEGF signaling during development is further evidenced by the lethal phenotype of mice that lack only one allele of the VEGF gene (Carmeliet et al. (1996) Nature 380:435-439; Ferrara et al. (1996) Nature 380:439-442) and also of mice that only express the smallest isoform of VEGF (VEGF-120) (Carmeliet et al. (1999) Nature Med. 5, 495-502). Thus, deviations on either side from a precisely determined level of VEGF signaling results in embryonic lethality.
Mice deficient in VEGFR2 lack both endothelial cells and hematopoietic cells, a more severe phenotype compared to that of VEGFR1 knockout, that results in embryonic lethality at day 8 (Shalaby et al. (1995) Nature 376:62-66). This is presumably a consequence of the fact that these two cell types arise from a common, VEGFR2-expressing precursor, the hemangioblast (Eichmann et al. (1997) Proc. Natl. Acad. Sci. 94: 5141-5146).
Structural Requirements for Binding
Crystal structure of the receptor-binding domain of VEGF (residues 8-109) has recently been reported (Muller et al. (1997) Proc. Natl. Acad. Sci., U.S.A. 94: 7192-7197; Muller et al. (1997) Structure 5:1325-1338). In the VEGF homodimer, the monomers are oriented in an antiparallel manner with two intersubunit disulfide bonds being formed between Cys51 from one subunit and Cys60 from the other. The three intrasubunit disulfide bonds are clustered in a characteristic cysteine knot motif (Sun et al. (1995) Annu. Rev. Biophys. Biomol. Struct. 24: 269-291) also observed in PDGF and TGFxcex22. Despite low sequence homology (about 20%), PDGF and VEGF have very similar structures. Both proteins have an elongated shape in which each of the subunits consist primarily of four antiparallel xcex2 strands connected with three solvent accessible loops. In the homodimer, loops I and III from one subunit are adjacent to loop II from the other subunit. Alanine-scanning mutagenesis studies of VEGF have identified discrete regions that are important for high affinity binding to VEGFR1 and VEGFR2 (Keyt et al. (1996) J. Biol. Chem. 271, 5638-5646; Muller et al. (1997) Proc. Natl. Acad. Sci., U.S.A. 94, 7192-7197). Amino acid residues most critical for binding of VEGF to VEGFR1 are D63 and E64 in loop II. Residues most critical for binding of VEGF to VEGFR2 are R82-H86 encompassing loop III, I46 in loop I and E64 in loop II. Knowledge of the importance of these regions for receptor binding has been utilized to generate VEGF mutants in which only one side of the VEGF homodimer was rendered defective for receptor binding (Siemeister et al. (1998) Proc. Natl. Acad. Sci., U.S.A. 95: 4625-4629; Fuh et al. (1998) J. Biol. Chem. 273: 11197-11204). As expected, such monovalent VEGF mutants are inhibitors of VEGF-induced signaling since they are deficient in their ability to dimerize the receptors. Interestingly, avidity effects play a greater role in the binding of VEGF to VEGFR2 than to VEGFR1. The affinity of monomeric VEGFR1 for wild-type VEGF dimer is reduced only about 2-fold compared to that of dimeric VEGFR 1 (IgG fusion construct) (Weismann et al. (1997) Cell 91, 695-704). In contrast, the affinity of monomeric VEGFR2 for VEGF is reduced 100-fold compared to the dimeric VEGFR2 (Fuh et al. (1998) J. Biol. Chem., ibid). Comparing only the monomeric forms, VEGFR1 binds to VEGF with about 100-fold higher affinity compared to VEGFR2.
Domain deletion studies of the extracellular region of the VEGF receptors have shown that out of seven IgG-like domains, domains 2 and 3 of VEGFR1 (Davis-Smyth et al. (1996) EMBO J. 15: 4919-4927; Barleon et al. (1997) J. Biol. Chem. 272: 10382-10388) and VEGFR2 (Fuh et al. (1998) J. Biol. Chem. 273, 11197-11204; Shinkai et al. (1998) J. Biol. Chem. 273, 31283-31288) are essential for VEGF binding. Crystal structure of the complex between VEGF8-109 with IgG domain 2 of VEGFR1 (that bind to VEGF with only 60-fold reduced affinity compared to the entire extracellular domain of the receptor) shows the receptor to be in contact with both subunits of VEGF8-109 in an interaction dominated by hydrophobic contacts (Weismann et al. (1997) Cell, ibid).
VEGF-165 Receptors
In addition to VEGFR1 and VEGFR2, receptors that only bind VEGF-165 and not VEGF-121 have been identified on endothelial cells and some tumor cells (Soker et al. (1996) J. Biol. Chem. 271: 5761-5767; Soker et al. (1997) J. Biol. Chem. 272: 31582-31588; Omura et al. (1997) J. Biol. Chem. 272: 23317-23322). One such receptor unrelated in sequence to the tyrosine kinase receptors and with a short cytoplasmic domain, neuropilin-1, is also a receptor for semaphorins which play a role in neuronal chemorepulsion during development (Soker et al. (1998) Cell 92:735-745). Since the binding of VEGF-165 to neuropilin-1 involves the exon 7-encoded domain that is not required for the binding to VEGFR1 and VEGFR2, it has been suggested that neuropilin-1 serves as a co-receptor for VEGF-165. The presence of such receptors on endothelial cells may in part account for the enhanced mitogenic activity of VEGF-165 compared to VEGF-121. Consistent with this notion is the observation that cardiovascular system of neuropilin-1 knockout mice does not develop normally leading to embryonic lethality (Kitsukawa et al. (1997) Neuron 19: 995-1005). The questions of what role VEGF may play in neuronal development and conversely, whether semaphorins have a role in vascular development and function, remain to be answered.
Receptor Binding Specificity of Various Forms of VEGF and Other Proteins in the VEGF Family
In addition to the alternatively spliced forms of VEGF, additional species can be generated by proteolytic processing. Plasmin cleaves VEGF-165 and VEGF-189 between residues Arg-110 and Ala-111 to generate VEGF-110 as the amino terminus fragment (Keyt et al. (1996) J. Biol. Chem., ibid; Plouxc3xat et al. (1997) J. Biol. Chem 272: 13390-13396). Since it contains the receptor binding domain (supra), VEGF-110 bind to both VEGFR1 and VEGFR2. Like VEGF-121, VEGF-110 does not bind to heparin and its potency is lower compared to that of VEGF-165 (Keyt et al. (1996) J. Biol. Chem., ibid). Interestingly, VEGF-189 can bind to VEGFR1 but not VEGFR2 and this renders it inactive as an endothelial cell mitogen (Houck et al. (1991) Mol. Endocrinol., ibid; Plouxc3xat et al. (1997) J. Biol. Chem. 272, ibid). VEGF-189 thus requires proteolytic processing either by plasmin or by urokinase-type plasminogen activator (that cleaves VEGF-189 in the exon 6encoded domain to generate a 40 kDa fragment) to gain ability to bind to VEGFR2 (Plouxc3xat et al. (1997) J. Biol. Chem., ibid).
Proteins with sequence homology to VEGF (also referred to as VEGF-A) have recently been described including placenta growth factor (PlGF: Park et al. (1994) J. Biol. Chem. 269: 25646-25654), VEGF-B (Olofsson et al. (1996) Proc. Natl. Acad. Sci., U.S.A. 93: 2576-2581), VEGF-C (Lee et al. (1996) Proc. Natl. Acad. Sci., U. S. A. 93:1988-1992; Joukov et al. (1996) EMBO J. 15: 290-298), VEGF-D (Achen et al. (1998) Proc. Natl. Acad. Sci., U.S.A. 95: 548-553) and VEGF-E (Ogawa et al. (1998) J. Biol. Chem. 273: 31273-31282). In terms of receptor binding specificity, PIGF and VEGF-B can bind only to VEGFR1 with high affinity. VEGF-C and VEGF-D bind to VEGFR2 and another related tyrosine kinase, Flt-4 or VEGFR3. The expression of VEGFR3 appears to be confined to lymphatic endothelial cells. VEGF-E, a protein encoded in the genome of the Orf virus, binds only to VEGFR2 (Ogawa et al. (1998) J. Biol. Chem. 273: 31273-31282). Some of these proteins including PIGF and VEGF-B can form heterodimers with VEGF (Cao et al. (1996) J. Biol. Chem. 271: 3154-3162; DiSalvo et al. (1996) J. Biol. Chem. 270: 7717-7723). The function of these VEGF-related molecules in physiological and pathological conditions remains to be precisely defined, however, it is clear that some redundancy of signaling mediated by VEGF receptors exists (Nicosia (1998) Am. J. Pathol. 153: 11-16).
VEGF Receptors on Non-endothelial Cells
Although VEGFR1 and VEGFR2 are expressed predominantly on endothelial cells, they have also been detected on some non-endothelial cells. VEGFR1 is expressed on trophoblasts (Charnockjones et al. (1994) Biol. Reprod. 51: 524-530), monocytes (Barleon et al. (1996) Blood, ibid), hematopoietic stem cells and megakaryocytes/platelets (Katoh et al. Cancer Res. 55: 5687-5692), renal mesangial cells (Takahashi et al. (1995) Biochem. Biophys. Res. Commun. 209: 218-226) and pericytes (Yamagishi et al. (1999) Lab. Invest. 79: 501-509). In monocytes, VEGFR1 is responsible for the VEGF-mediated induction of migration and tissue factor expression (Clauss et al. (1996) J. Biol. Chem., ibid; Barleon et al. (1996) Blood, ibid; Hiratsuka et al. (1998) Proc. Natl. Acad. Sci., ibid). In pericytes, VEGFR1 may mediate the recently described ability of VEGF to act as a mitogen and chemotactic factor (Yamagishi et al. (1999) Lab. Invest., ibid). The role of VEGFR1 in trophoblasts and mesangial cells remains to be elucidated. The expression of VEGFR2 has been detected on hematopoietic stem cells, megakaryocytes/platelets and retinal progenitor cells (Katoh et al. (1995) Cancer Res. 55: 5687-5692; Yang et al. (1996) J. Neurosci. 16: 6089-6099). VEGFR1 and VEGFR2 expression has also been reported on malignant cells including leukemia cells (Katoh et al. (1995) Cancer Res., ibid) and melanoma cells (Gitay-Goren et al. (1993) Biochem. Biophys. Res. Commun. 190: 702-709).
SELEX
A method for the in vitro evolution of nucleic acid molecules with high affinity binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichmentxe2x80x9d, now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled xe2x80x9cNucleic Acid Ligandsxe2x80x9d, now U.S. Pat. No. 5,475,096, and U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled xe2x80x9cMethods for Identifying Nucleic Acid Ligandsxe2x80x9d, now U.S. Pat. No. 5,270,163 (see also WO91/19813), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describe a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield high affinity nucleic acid ligands to the target molecule.
The basic SELEX method may be modified to achieve specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled xe2x80x9cMethod for Selecting Nucleic Acids on the Basis of Structurexe2x80x9d, now abandoned, describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA (See U.S. Pat. No. 5,707,796). U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled xe2x80x9cPhotoselection of Nucleic Acid Ligandsxe2x80x9d, now abandoned, describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled xe2x80x9cHigh-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeinexe2x80x9d, now abandoned, describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed xe2x80x9cCounter-SELEXxe2x80x9d (See U.S. Pat. No. 5,580,737). U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Solution SELEXxe2x80x9d, now abandoned, (See also U.S. Pat. No. 5,567,588) and U.S. patent application Ser. No. 08/792,075, filed Jan. 31, 1997, entitled xe2x80x9cFlow Cell SELEXxe2x80x9d, now U.S. Pat. No. 5,861,254 describe SELEX-based methods which achieve highly efficient partitioning between oligonucleotides having high and low affinity for a Target molecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992, entitled xe2x80x9cNucleic Acid Ligands to HIV-RT and HIV-1 Revxe2x80x9d, now U.S. Pat. No. 5,496,938, describes methods for obtaining improved Nucleic Acid Ligands after the SELEX process has been performed. U.S. patent application Ser. No. 08/400,440, filed Mar. 8, 1995, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEXxe2x80x9d, now U.S. Pat. No. 5,705,337 describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or delivery. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. Specific SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled xe2x80x9cHigh Affinity Nucleic Acid Ligands Containing Modified Nucleotidesxe2x80x9d, now abandoned, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2xe2x80x2-positions of pyrimidines, as well as specific RNA ligands to thrombin containing 2xe2x80x2-amino modifications (See U.S. Pat. No. 5,660,985). U.S. patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2xe2x80x2-amino (2xe2x80x2-NH2), 2xe2x80x2-fluoro (2xe2x80x2-F), and/or 2xe2x80x2-O-methyl (2xe2x80x2-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled xe2x80x9cNovel Method of Preparation of Known and Novel 2xe2x80x2 Modified Nucleosides by Intramolecular Nucleophilic Displacementxe2x80x9d, now abandoned, describes oligonucleotides containing various 2xe2x80x2-modified pyrimidines. PCT/US98/00589, filed Jan. 7, 1998, entitled xe2x80x9cBioconjugation of Oligonucleotidesxe2x80x9d (WO 98/30720) describes a method for identifying bioconjugates to a target comprising nucleic acid ligands derivatized with a molecular entity exclusively at the 5xe2x80x2-position of the nucleic acid ligands.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEXxe2x80x9d, now U.S. Pat. No. 5,637,459 and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Blended SELEXxe2x80x9d, now U.S. Pat. No. 5,683,867 respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules. The full text of the above described patent applications, including but not limited to, all definitions and descriptions of the SELEX process, are specifically incorporated herein by reference in their entirety.
The present invention includes methods of identifying and producing nucleic acid ligands to a VEGF receptor and the nucleic acid ligands so identified and produced. A VEGF receptor is any receptor which VEGF binds, including, but not limited to, VEGFR1, VEGFR2, VEGFR3, neuropilin-1. By substantially homologous it is meant a degree of amino acid sequence identity of 70% or more. In particular, RNA sequences are provided that are capable of binding specifically to a VEGF receptor. Also included are oligonucleotides containing nucleotide derivatives modified at the 2xe2x80x2 position of the pyrimidines. Specifically included in the invention are the RNA ligand sequences shown in Tables 2 and 3 and FIG. 1 (SEQ ID NOS:2-36). Also included in this invention are RNA ligands of a VEGF receptor that inhibit the function of VEGF signaling.
Further included in this invention is a method of identifying nucleic acid ligands and nucleic acid ligand sequences to a VEGF receptor, comprising the steps of (a) preparing a candidate mixture of nucleic acids, (b) contacting the candidate mixture of nucleic acids with a VEGF receptor, (c) partitioning between members of said candidate mixture on the basis of affinity to a VEGF receptor, and (d) amplifying the selected molecules to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity for binding to a VEGF receptor.
More specifically, the present invention includes the RNA ligands to a VEGF receptor, identified according to the above-described method, including those ligands shown in Tables 2 and 3 and FIG. 1 (SEQ ID NOS:2-36). Also included are nucleic acid ligands to a VEGF receptor that are substantially homologous to any of the given ligands and that have substantially the same ability to bind a VEGF receptor and inhibit VEGF signaling. Further included in this invention are nucleic acid ligands to a VEGF receptor that have substantially the same structural form as the ligands presented herein and that have substantially the same ability to bind a VEGF receptor and inhibit VEGF signaling.
The present invention also includes other modified nucleotide sequences based on the nucleic acid ligands identified herein and mixtures of the same.